Crystal structures of human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating protein (RabGAP) domains reveal critical elements for GLUT4 translocation

Abstract

We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 Å resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 α-helices and no β-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met(930), corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met(930) and Leu(1019) predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.

FIGURE 1.

Superimposed structures of TBC1D1, TBC1D4, and Gyp1p RabGAP domains. A, schematic ribbon diagrams of the superimposed TBC1D1 (orange) and TBC1D4 (gray) RabGAP domain structures are shown with catalytic residues and α-helices labeled. The structures are highly similar (C_α r.m.s.d. = 0.865 Å), with minor differences within the loops connecting the amino-terminal three helices. B, structures of the yeast Gyp1p (lavender), mouse Rab33B (green), GDP (yellow) complex (PDB 2G77), with TBC1D1 (orange) are superimposed on Gyp1p.

FIGURE 2.

Structure-based sequence alignment of Gyp1p, TBC1D1, and TBC1D4 RabGAP domains. Alignments are according to common structural elements (light blue boxes) and protein sequence. Disordered regions are denoted by yellow boxes, asterisks indicate catalytic residues, and dots denote gaps in sequence relative to other domains. Gyp1p residues important for Rab33B binding via main chain and side chain interactions are shaded green and red, respectively.

FIGURE 3.

Structural comparisons among TBC1D1, TBC1D4, and Gyp1p RabGAP domains. A, significant differences between Gyp1p and TBC1D1 or TBC1D4 are the residues between Gyp1p α7 and α8, creating α7a and α7b, which are absent in TBC1D1 and TBC1D4. The space occupied by these short helices in Gyp1p is occupied in TBC1D1 and TBC1D4 by the alternative ancillary helix α1′. B, α4 in Gyp1p is also much longer than in the TBC1D domains. Much of this region in TBC1D1 and TBC1D4 is a disordered loop.

FIGURE 4.

Catalytic activities of TBC1D1 RabGAP domains. A, TBC1D1 and TBC1D4 RabGAP domains were used to catalyze hydrolysis of Rab14-loaded GTP. For each RabGAP domain concentration, k_obs was fitted using a pseudo first-order Michaelis-Menten model (A(t) = (A_∞ − A₀) (1 − exp(−k_obst)) + A₀). B, the catalytic efficiency parameter, K_cat/K_m, was calculated by plotting k_obs against TBC1D1 concentration, and the slopes of the best fit lines provide k_cat/K_m values.

FIGURE 5.

Lack of sequence similarity at Rab binding surfaces of TBC1D1 and Gyp1p. A, Rab binding surfaces predicted by the Gyp1p/Rab33B structure are poorly conserved between TBC1D1 and Gyp1p. B, because the sequence conservation is so low, we predicted additional solvent-exposed residues of TBC1D1 (Pro⁹²⁸, Met⁹³⁰, and Glu⁹⁵⁹) that might participate in Rab binding.

FIGURE 6.

Sequence alignment of mammalian Rab proteins at the predicted RabGAP interface. The buried region of mouse Rab33B at the yeast Gyp1p interface was aligned with other human Rab proteins reported potentially to interact with TBC1D proteins during GLUT4 trafficking. Rab residues important for Gyp1p RabGAP binding via main chain and side chain interactions are colored green and red, respectively.

FIGURE 7.

Assay results for catalytic activity and GLUT4 translocation. A, relative k_cat/K_m values for RabGAPs against GTP-loaded Rab14 are plotted relative to WT protein k_cat/K_m. B, L6 muscle cells expressing a myc-GLUT4-GFP reporter were transfected with plasmids expressing WT or substituted mouse TBC1D1 proteins (numbering for the human protein provided for comparison). *, p < 0.05; ***, p < 0.0005.

FIGURE 8.

GLUT4 translocation assay. A, equivalent expression of the TBC1D1 proteins was verified by Western blotting lysates from the transfected L6 myocytes using an anti-HA antibody. B, single-cell fluorescence assay shows co-expression of myc-GLUT4-GFP and HA-TBC1D1. C, differential effects of the HA-TBC1D1 proteins on insulin-induced myc-GLUT4-GFP translocation in L6 myocytes are shown. DAPI stains nuclei blue; Myc-GLUT4-GFP fluorescence is red due to binding of Cy3-conjugated secondary antibody.